Determining how combinations of genes interact as gene regulatory networks to produce cellular diversity is fundamental to understanding development. The neural crest (NC) has been studied extensively to elucidate mechanisms of cell diversification during development. The NC is a discrete and seemingly homogeneous undifferentiated stem cell-like ectodermal population of vertebrate embryonic precursor's cells that is the source of multiple different cell types including neurons and glia of the peripheral nervous system, pigment cells and major elements of the craniofacial skeleton, among others. Subsequent to the induction of the NC domain of the ectoderm during gastrulation, the fates of subsets of NC cells are specified as distinct sublineages that ultimately generate the complete cellular derivative repertoire of the progenitor population. How the fates of NC sublineages are specified during development is incompletely understood. Determining at the genetic level how differences between NC cells are established is essential to understanding how the NC generates such a vast array of different cell types. Studies in zebrafish and other vertebrates have indicated that several transcription factors are essential for the specification of distinct and overlapping subsets of NC sublineages, although none can individually account for NC cell diversification in its entirety. We found that in zebrafish foxd3; tfap2a double mutants all NC sublineages fail to be specified, indicating that foxd3 and tfap2a are synergistically and universally required for the initiation of NC diversification. Further, our studies indicate that the requirement for foxd3 and tfap2a for the initial specification of NC sublineages is due in part to their regulation of the NC expression of the SoxE family genes sox9a, sox9b and sox10. Together, these results have identified a framework gene regulatory network (GRN) that initiates NC diversification. Critically, however, the mechanisms by which framework GRN transcription factor interactions initiate NC diversification are not known. Equally important, the identified framework GRN cannot account for NC diversification in its entirety. Accordingly, we propose a research plan, based on the established framework GRN, to answer critical unresolved questions about the genetic regulation of the specification of NC sublineage fates which ultimately produces NC diversity. We will determine at the molecular level, employing a ChIP-based approach coupled with transgenic reporters, the mechanisms by which interactions between the frameworks GRN transcription factors specify NC cell fates. In addition, we will comprehensively identify additional foxd3- and tfap2adependent genes that, based on selection criteria, are candidates for the GRN controlling NC diversification using whole genome microarray expression profiling. We will then determine the functions of these candidates in regulating NC diversification using loss- and gain-of function approaches employing transgenic reporter wild type embryos and embryos singly or doubly mutant for genes comprising the framework GRN (foxd3, tfap2a, sox9a, sox9b and sox10) coupled with comprehensive phenotypic analysis of NC development. The results of our proposed studies will address critical deficiencies in the field by producing major fundamental advances in our understanding of the regulation of NC diversification. In addition, our results will generate applicable mechanistic paradigms for understanding cell diversification generally and provide a rich foundation for future comprehensive functional determination of the complete GRN controlling NC development. Lastly, given the high prevalence of clinically relevant conditions resulting from miscues during NC development, our results are likely to provide important insights for strategies to diagnose, treat and prevent human diseases such as neurocristopathies and cancers of NC origin. PUBLIC HEALTH RELEVANCE: The process by which unspecialized cells of early embryos go on to generate vast numbers of extremely different cell types specialized to subserve the diverse functional requirements of the developing organism is referred to as cell diversification. To understand how this important process is regulated at the level of genes and combinations of genes working together, it is useful to study less intricate model systems compared to whole organisms. The vertebrate neural crest has been studied extensively as a model to learn how cell diversification occurs because, during embryonic development, this population of seemingly homogeneous embryonic precursor cells ultimately generates an extensive array of different cell types contributing to multiple organ systems. While a great deal has been learned about the genetic regulation of neural crest diversification, much remains incompletely understood. Therefore, the purpose of our proposed research is to utilize the neural crest to experimentally determine at the molecular level how as yet poorly understood aspects of cell diversification are regulated during development. The results of our proposed research will provide both critical new mechanistic insights into the development of the neural crest and inform our understanding of how embryonic cell diversification is regulated generally. In addition, as cells and tissues derived from the neural crest are essential for the function and survival of vertebrate organisms and miscues during neural crest development are known to underlie large numbers of diseases and other clinically relevant conditions in humans, our research will provide insights into how these diseases develop and how they can be ameliorated. Further, not only is the neural crest the sources of prominent cancers, many genes that control the processes underlying neural crest diversification are implicated in cancer development and metastasis more generally. Therefore, taken together, our proposed research will also contribute to the development of strategies for the diagnosis, treatment and prevention of human diseases.